The PTEN tumor suppressor gene is mutated, deleted or expressed at reduced levels in a large proportion of human cancers. PTEN is a lipid and protein phosphatase that negatively regulates phosphoinositol-3-kinase (PI3K)/AKT signaling by dephosphorylating phosphatidylinositol-3, 4, 5-triphosphate (PIP3). Unrestrained PI3K/AKT signaling leads to increased cell proliferation and reduced cell death, thereby driving tumorigenesis. Although antagonizing PI3K/AKT signaling is considered the primary physiological role of PTEN and its most relevant property as a tumor suppressor, PTEN may have additional tumor suppressive functions, for instance as a guardian of structural chromosome integrity. However, there is an incomplete understanding of the full repertoire of the normal and tumor suppressive PTEN functions and the extent to which PTEN mutations found in human cancer affect each of these functions. This represents a critical barrier that is slowing down progress toward improving treatment strategies for a large segment of cancer patients. We provide preliminary data that point to a novel biological function of PTEN in ensuring accurate chromosome segregation in mitosis and maintaining chromosome number stability. The central objective of this application is to decipher how mechanistically PTEN regulates proper chromosome segregation, and to determine the extent to which loss of this function contributes to malignant cell transformation, with the ultimate goal to exploit this knowledge for preventive and therapeutic purposes. Our central hypothesis is that PTEN, through its PDZ-interaction domain, regulates proper chromosome segregation in mitosis and that this novel function represents a critical tumor protective function of PTEN. We will test this hypothesis by pursuing two specific aims. In the first aim, we will determine the mechanism by which PTEN regulates proper chromosome segregation by using mouse embryonic fibroblasts (MEFs) from Pten+/- and other mutant mouse strains in combination with a comprehensive set of cell biological and biochemical approaches. In the second aim, we will establish the role of the Pten PDZ- interaction domain in normal development and tumor suppression using a newly generated knockin mouse strain that lacks this domain. We will determine the mitotic, developmental and cancer phenotypes of mice that are heterozygous and homozygous for this knockin allele and compare these to those of Pten+/- mice. The expected overall impact of this innovative proposal is that it will fundamentally advance our mechanistic understanding of the normal and neoplastic functions of the second most frequently mutated tumor suppressor gene in human cancer. This knowledge will lay the foundation for development of new therapeutic strategies that will improve the clinical outcome of cancer patients with alterations in PTEN, in addition to conceptually advancing the fields of mitosis and cancer biology.